This invention relates to hematology control compositions, and methods for their use in a reference standard for particle analysis instrumentation of the COULTER.RTM. type. More particularly, this invention relates to a three-component system for simulating the three major components of human leukocytes, namely lymphocytes, mononuclear cells and granulocytes, and optionally sub-divisions thereof, which is compatible with known red blood cell and platelet controls.
Mononuclear cells are single nucleated blood cells which include monocytes and numerous mature and immature forms of lymphocytes and immature myelocytes and erythrocytic blood cells. The modified COULTER COUNTER Model S Plus can deliniate mature lymphocytes and polymorphic granuloctyes from the general group of mononuclear cells found in circulating blood under normal and pathological conditions. These circulating mononuclear blood cells include promyelocytes, myelocytes and blast cells as well as monocytes. Since monocytes are the most prevalent mononuclear cell population under normal hemopoietic conditions, the 90 to 160 fL region can be referred to as the monocyte region.
The use of electronic particle counters in hematology is well known. U.S. Pat. No. 2,656,508 discloses a basic apparatus utilizing the Coulter principle for this purpose. U.S. Pat. No. 3,757,213 contains a description of several such devices which incorporate threshold circuitry. Threshold circuitry excludes random amplitude signals caused by noise and background debris of inconsequential particles in the suspension. It may also be used to limit the size range of the particles counted. Adjustable threshold circuits with dials marked off in mathematically related dial settings are in common use.
Although this disclosure will be directed primarily to embodiments involving the use of electronic particle counters of the COULTER type, it should be understood that the particle controls herein disclosed, and their methods of use described herein, find wide application with particle counters generally. Accordingly, the term "electronic particle counter" should be understood to include, in addition to COULTER COUNTER.RTM. instruments, any other type of particle counter which discriminates between particles of various sizes by the use of electronic discriminator circuits ("thresholds") which respond electronically to signals indicative of particle size, mass or particle volume. COULTER and COULTER COUNTER are Registered Trademarks of Coulter Electronics, Inc.
Calibration check techniques for red blood cell (erythrocyte), white blood cell (leukocyte) and platelet (thrombocyte) counts and physical attributes are well developed. The material used for checking calibration, hereinafter called a control, also can be used to calibrate a hematology instrument. The techniques for using a control generally involve counting known populations of particles suspended in a liquid vehicle in the control preparation, which usually is diluted substantially with a diluent prior to counting. Heretofore, however, no control had been developed for use with three sub-groups of leukocytes, namely, lymphocytes, mononuclear cells and granulocytes, since the equipment for automatic counting of these sub-groups had not been developed.
It is evident that a control product must accurately indicate on a comparative basis what a test sample of fresh blood constitutes with regard to the determinations in question. It is further evident how important it is for the control product to simulate fresh blood, since blood components, such as red blood cells, can hemolyze slowly and undergo changes in size and shape within hours after removal from a blood donor. Similarly, white blood cells suffer degenerative changes.
Quality control long has been a necessary and routine procedure in clinical hematology. Accuracy in the counting of red and white blood cells and in the making of hematocrit and hemoglobin determinations of the patient's serum is dependent, in part, upon the use of adequate control standards. Thus, the accuracy of the manual technique of particle counting, such as by the classical method of microscopy, can be checked by giving the technician a so-called "blind" sample, or control standard, containing a known concentration of particles for comparison with the unknown samples for which determination has to be made. With the numerous types of automatic equipment for particle counting now available, quality control by the use of control standards is likewise necessary since the possibility of malfunctioning of the instrument is ever present. Consequently, the importance of accurate and reliable checks on hematological determinations that may be used in the diagnosis of disease is clear. The traditional method of maintaining a quality control program for automatic particle counting equipment has consisted of providing fresh human blood as a whole blood standard. However this fresh blood is usable for only one day. Consequently, durable blood products were developed which did not require fresh human blood.
In copending application Ser. No. 454,926, filed Jan. 3, 1983, now U.S. Pat. No. 4,485,175 an automatic method is described for differential determination of three populations of leukocytes using a COULTER COUNTER Model S Plus automated blood counter. Accordingly there is now a need for a reliable check of threshold calibration and additional operational performances for electronic particle counters typified by the COULTER COUNTER analyzers now marketed. Operators then can identify and document the volume ranges between which the three populations of leukocytes are to be counted routinely. There is further a need in the art for a reliable method for demonstrating instrument stability over a prolonged period of use.
Human lymphocytes, mononuclear cells and granulocytes have a specific size distribution range and after stabilization (for example with a fixative, such as formaldehyde), their responsiveness in diluents may not permit proper size discrimination. This would result in an inability to evaluate proper instrument operation. Both the upper and lower size limits for each population of leukocytes must be represented in the reference control material. In addition, the mean cell volume of each leukocyte population in the reference control material should be very close to that of normal human blood. When upper and lower size limits and mean cell volume are thus specified, it becomes a virtual necessity for the volume distribution histogram of the control material to approximate the normal distribution of the fresh human cells. This volume distribution must remain relatively constant regardless of the total white cell count and the range of ratios for all three populations representing abnormal low, normal and abnormal high conditions for human leukocytes. Therefore, it is necessary that the preservation process used in the manufacture of the reference control suspension does not cause significant shrinking or swelling of the cells. Also one must be sure that aging of the reference control does not result in deterioration of the volume distribution histogram characteristics or other parameters. A further requirement for the leukocyte component in a whole blood reference control for multi-parameter instruments is that the cells must not be completely lysed by the lytic reagent.
With the increasing use of automated devices capable of performing multiple hematological determinations and with the introduction of techniques of automated cell counting, the solution to the problem lies not in the pursuit of more effective ways of stabilizing "real" human leukocytes, but in substituting a surrogate which satisfies the specifications against which the product is made. Therefore, animal cells which could be converted into a useful control or calibrator were sought.
Numerous citations to prior art are set forth with some explanation in this application. To the extent that further information regarding such prior art might be needed to more fully understand the herein invention, the following are incorporated by reference: U.S. Pat. Nos. 4,179,398; 4,213,876; 4,264,470; 4,299,726; 4,389,490; 4,405,719 and U.S. Application Ser. No. 454,926, filed Jan. 3, 1983 now U.S. Pat. No. 4,485,175.